As is well known in the art, genetic information is encoded on double stranded DNA molecules according to the sequence of four nucleotides containing different bases, adenine (A), thymine (T), cytosine (C) and guanine (G). Blocks of DNA sequences flanking genes often control gene activity by binding regulatory proteins and acting as recognition signals for enzymes of the cells biosynthetic machinery. Thus each cell contains a web of regulatory molecules which, by binding to specific DNA sequences, control gene activity. Other DNA sequences have crucial functions related to the control of DNA synthesis and partitioning of DNA into separate cells during cell division. These functions must be present on every DNA molecule in every cell or the DNA will be lost within a few cell generations.
Plasmids are usually circular DNA molecules possessing DNA sequences allowing them to replicate independently from chromosomal DNA. The DNA sequence block where the replication of plasmid DNA is initiated is commonly called the "origin of replication" and the ability to replicate independently from chromosomal DNA is referred to as "extrachromosomal" replication.
Molecular biologists have developed techniques for cutting DNA molecules into fragments using sequence specific restriction enzymes, purifying the fragments and rejoining them in a different order. If one of the fragments of DNA used contains an origin of replication from an E. coli plasmid, the DNA can be inserted (transformed) into E. coli where it will replicate as a plasmid and can be produced in relatively large quantities. These techniques mean that genes from one organism, for example a human gene, can be flanked by regulatory DNA sequences from another organism, for example the bacterium E. coli, causing the human gene to be active in E. coli under entirely different regulatory controls. If the plasmid in question is constructed to include a second origin of replication allowing replication in a separate host cell, for example a mouse cell line, the gene can easily be transferred to the second host cell. Such a plasmid containing origins of replication for more than one host is commonly called a "shuttle vector". Plasmids are usually constructed to contain selectable markers, which are usually genes that confer antibiotic resistance or a metabolic advantage on the host cell to allow cells containing the plasmid to be distinguished from cells that have not received any plasmid during the transformation. Selectable marker genes must be flanked by appropriate DNA sequences to permit gene activity in the required host cell. It is possible to insert a plasmid into a host cell where it will be unable to replicate and so the only cells that survive the selection procedure will be those with the plasmid inserted into the host's chromosomal DNA. Such a plasmid without an appropriate origin of replication is called an "integrating plasmid".
A cell produces polypeptides and proteins by initially making a messenger RNA copy of the gene, a process called transcription which is under the control of the flanking DNA sequences as summarised above. The cellular biosynthetic machinery then reads (translates) the RNA sequence in three nucleotide groups called codons which specify the amino acids to be incorporated into the polypeptide chain. The genetic code and mechanism of protein synthesis is very similar in all organisms so molecular biology techniques can be used to construct plasmid vectors to produce recombinant proteins in many different host cells irrespective of the source of the original gene. However, different host cells may process the protein in different ways so it may, for example, be folded incorrectly or cleaved by protease enzymes. Most importantly, eukaryotic cells differ from bacteria by frequently linking further chemical structures onto their proteins, a process called "post-translational modification". The chemical structures linked to eukaryotic proteins may include several types of oligosaccharide chains, glycolipids, lipids, sulphate and phosphate groups, all of which may affect the physical and biological properties of the molecule. Common effects of these post-translational modifications include increased resistance to proteolysis, altered immunogenicity, altered in vivo clearance and uptake by different cell types.
Post-translational modifications frequently occur on proteins that are secreted from cells or are present on cell membranes. Such proteins include a wide variety of soluble proteins that mediate inter-cellular interactions, blood proteins and cell surface receptors and so are of considerable interest to the pharmaceutical industry as either the targets for drug research or for in vivo administration as therapeutic drugs in their own right. Since post-translational modifications may substantially alter the biological activity of such proteins (for example, tissue plasminogen activator (Ezzell, 1988, Nature 333, 383)), it is a goal of the biotechnology industry to produce each protein with a range of different modifications, both those that occur naturally and new modifications such as truncated oligosaccharide chains. However, proteins with post-translational modifications can only be produced in eukaryotic hosts and only a few eukaryotes have been used industrially. Mammalian tissue culture, for example Chinese Hamster Ovary Cells, is usually able to produce proteins with post-translational modifications similar to the natural protein, but is very expensive since these cells frequently require serum components in their growth media, have a slow growth rate and are relatively difficult to grow in large fermentors. Consequently, simple eukaryotes such as insect cells infected with baculovirus or yeast cells have been used to produce proteins with some post-translational modifications at a considerably lower cost. However, no one host is suitable for all recombinant proteins or can produce more than a few of the wide range of desirable post-translational modifications.
Dictyostelium has some advantages as a host for the production of low cost recombinant proteins with post-translational modifications (reviewed by Glenn & Williams, 1988, Australian J. Biotech. 1(4), 46-56). These include the production of N-linked gycosylation indistinguishable from the mammalian "high mannose form" and a wide variety of other structures including phosphatidyl-inositol-glycan tails. It is possible to alter the post-translational modifications produced by Dictyostelium by either using a range of mutant cultures which produce altered glycan structures or by simply harvesting the Dictyostelium cells at different stages of the life cycle. A considerable body of scientific literature is available on the culture and genetics of Dictyostelium (Spudich J. Ed. (1987) Methods in Cell Biology Vol. 28, Academic Press, London). Dictyostelium has a number of characteristics suitable for use in the production of recombinant proteins in fermenters since they grow rapidly (4-10 hour cell cycle) and reach high densities (around 50 million cells per ml) in a nutrient medium. For some purposes, the ability of Dictyostelium to grow on a lawn of bacteria on a simple nutrient medium provides a remarkably simple and cheap culture technique when compared with mammalian or event insect tissue culture.
Dictyostelium strains are known to posses at least thirteen different plasmids (Farrat & Williams (1988) Trends in Genetics 4, 343-348), but only Ddp1, Ddp2 and pDG1 have been studied in detail. Plasmid pDG1 is very unstable when cloned in E. coli (Orii et al (1989) Nucleic Acids Research 17, 1395-1408) so most constructions of shuttle vectors have used sequences from either Ddp1 or Ddp2. Plasmid Ddp1 is 12.3 Kb in size, but Ahern et al (Nucleic Acids Research (1988) 16, 6825-6837) showed that a vector containing a selectable marker (G418) resistance and only 2.2 Kb of Ddp1 was able to replicate extrachromosomally in D. discoideum. However, but the copy number per cell of this truncated plasmids lowered from the 150 characteristic of the parent plasmid to only 10-15 copies per cell. It is probable that this low copy number plasmid may not segregate efficiently at cell division and so may be unstable in the absence of continuous selection with the antibiotic G418. Incorporation of additional Dictyostelium DNA into such plasmids based on the Ddp1 origin of replication prevents them being maintained extrachromosomally (Gurniak et al, (1990) Current Genetics 17, 321-325.) so they are unsuitable for use in the biotechnology industry.
The practical application of plasmids constructed from sections of Ddp2 has been limited by technical difficulties. The majority of techniques used in molecular biology are designed for use in the bacterium E. coli so the manipulation of Dictyostelium DNA requires it to be cloned into a vector capable of replication in E. coli. Consequently, research on Ddp2 has concentrated on the construction of recombinant "shuttle vectors" containing sequences allowing replication in both E. coli and Dictyostelium spp. Plasmid pMUW111 illustrates a shuttle vector that the present inventors have constructed (FIG. 4), which contains a 4.139 Kb Hind III--ScaI restriction fragment of Ddp2. This is close to the minimum amount of Ddp2 which can maintain extrachromosomal replication in wild type strains of Dictyostelium. Leiting and Noegel (1988 Plasmid 20, 241-248) have used a similar 4.0 Kb fragment of Ddp2 with approximately 300 bp deleted close to the Xho I restriction site to construct a 9.6 Kb shuttle vector called pnDe1. However, despite containing minimal sections for the extrachromosomal replication of Ddp2, both these shuttle vectors (pMUW111 and pn DE1) suffer from problems of instability when maintained in E. coli. This is consistent with the Ddp2 DNA containing sequences that are unstable in E. coli. This problem can be mitigated by the use of host strains which lack exo-nuclease I and have low plasmid copy number (e.g. strain CES 201), but such hosts frequently present problems in preparing sufficient plasmid DNA for gene cloning experiments and for transforming back into Dictyostelium.
The necessity of using pieces of Ddp2 DNA approximately 4 Kb long to construct shuttle vectors also raises problems with regard to the final size of the plasmid. The shuttle vector must contain selectable markers for both hosts together with appropriate promoter and termination sequences. These sequences comprise nearly 50% of the size of plasmids pMUW111 and pnDe1. In addition, to be of any practical use a shuttle vector must be capable of carrying additional DNA containing a gene to be expressed in Dictyostelium together with appropriate controlling sequences. These additional sequences are likely to amount to a minimum of at least 2 Kb of DNA, bringing the total plasmid size to around 12 kilobase pairs. Increasing the size of the plasmid to over 10 Kb decreases its stability, a factor of considerable importance for the commercial production of recombinant proteins where, in order to avoid contamination of the product, regulatory authorities do not permit the use the antibiotic selection to ensure plasmid maintenance while cells are grown for extended periods. A large plasmid also raises difficulties since fewer restriction enzymes will cut the plasmid at only one position, the most suitable sites for genetic manipulations.
Shuttle vectors capable of being easily manipulated in E. coli and transferred back into Dictyostelium spp. are an essential pre-requisite for realising the potential of Dictyostelium in biotechnology. The present inventors have discovered means by which such vectors containing sections of Ddp2 smaller than 4 Kb can be constructed.
The present inventors have elucidated the full nucleotide sequence of the plasmid Ddp2 and have determined that a portion of this sequence encodes a gene designated Rep. The present inventors have shown that the presence of a polypeptide encoded by the Rep gene is essential for extrachromosomal replication of the Ddp2 plasmid.